A mass spectrometry‐based non‐radioactive differential radial capillary action of ligand assay (DRaCALA) to assess ligand binding to proteins

Abstract Binding of ligands to macromolecules changes their physicochemical and enzymatic characteristics. Cyclic di‐GMP is a second messenger involved in motility/sessility and acute/chronic infection life style transition. Although the GGDEF domain, predominantly a diguanylate cyclase, represents one of the most abundant bacterial domain superfamilies, the number of cyclic di‐GMP receptors falls short. To facilitate screening for cyclic di‐nucleotide binding proteins, we describe a non‐radioactive, matrix‐assisted laser desorption and ionization time‐of‐flight (MALDI‐TOF)‐based modification of the widely applied differential radial capillary action of ligand assay (DRaCALA). The results of this assay suggest that the diguanylate cyclase/phosphodiesterase variant YciRFec101, but not selected catalytic mutants, bind cyclic di‐GMP. Highlights Cyclic di‐nucleotides are ubiquitous second messengers in bacteria. However, few receptors have been identified. Previous screening of cell lysates by differential radial capillary action of ligand assay (DRaCALA) using radioactive ligand identified cyclic di‐nucleotide binding proteins. A MALDI‐TOF‐based DRaCALA was developed to detect cyclic di‐nucleotide binding as a non‐radioactive alternative. Known cyclic di‐GMP binding proteins were verified and potential cyclic di‐GMP binding proteins were identified.

modification of the widely applied differential radial capillary action of ligand assay (DRaCALA). The results of this assay suggest that the diguanylate cyclase/ phosphodiesterase variant YciR Fec101 , but not selected catalytic mutants, bind cyclic di-GMP.

Highlights
• Cyclic di-nucleotides are ubiquitous second messengers in bacteria. However, few receptors have been identified.
• Previous screening of cell lysates by differential radial capillary action of ligand assay (DRaCALA) using radioactive ligand identified cyclic di-nucleotide binding proteins.
• A MALDI-TOF-based DRaCALA was developed to detect cyclic di-nucleotide binding as a non-radioactive alternative.
• Known cyclic di-GMP binding proteins were verified and potential cyclic di-GMP binding proteins were identified.

| INTRODUCTION
Binding of small molecules to macromolecules such as proteins has a substantial physiological impact as it alters the physicochemical properties and the functionality of the protein including modulation of enzymatic parameters through allosteric regulation and change in protein-protein interactions due to binding-induced conformational changes. 1 There are a variety of directed and unbiased experimental approaches to determine binding of small molecules to proteins and to assess their binding affinity. 2 Traditional biochemical approaches require specific purification of individual proteins including protocol optimization and therefore are not amenable for high-throughput screening. One of the most successful experimental approaches suitable for screening of a large number of candidate receptors for binding with candidate small ligands is the differential radial capillary action of ligand assay (DRaCALA). This screening assay does not require purification of the expressed protein but uses whole bacterial cell lysates containing plasmid-expressed candidate gene products. [3][4][5][6][7] Conduction of assay involves spotting mixtures of lysates with small ligands onto a dry nitrocellulose membrane. Small ligands that are mobile on nitrocellulose will migrate uniformly away from the initial site of application. In contrast, spotting extract expressing proteins that bind the small ligand will sequester the ligand at the site of application on the nitrocellulose membrane where the proteins are immobilized. Therefore, the distribution of the ligand in these two zones allows determination of the protein-ligand interaction. Initial approaches utilized radiolabeled ligands that can be detected using phosphorimager screens. The scans of exposed DRaCALA spots provide a visual image that reflects the quantitative analysis.
Cyclic di-nucleotides are ubiquitous second messengers in bacteria; however, the abundance of cyclic di-GMP turnover proteins is still not reflected by the number of identified receptors. 8 DRaCALA has been frequently applied to identify receptors for cyclic di-nucleotides, in particular, cyclic di-GMP and cyclic di-AMP, which are ubiquitous second messengers in Gram-negative and Gram-positive bacteria. 9,10 Thereby, lysates of bacterial libraries containing all plasmid-expressed gene products of an organism have been screened for cyclic dinucleotide binding proteins. With this experimental approach, receptors involved in, for example, regulation of cellulose biosynthesis such as BcsE, mannose-sensitive hemagglutinin ATPase MshE, and maintenance of osmohomeostasis and potassium transport have been identified. 4,11,12 A radioactive ligand, as in the originally described approach, needs to be prepared. However, radiolabeling is not readily applicable to every small molecular compound. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a universal detection modality that can be used to detect small molecules.
We have applied MALDI-TOF MS to detect binding of a ligand to a protein receptor on the nitrocellulose membrane. Thereby, assessment of binding can be achieved by measuring the ligand concentration at the application spot and at the edge of the larger spot area that the liquid reaches by capillary action (Figure 1).
To perform this assay, a single colony of the Escherichia coli binding by the application of 32 P cyclic di-GMP was essentially performed as described previously using the same plasmid constructs. 3,7 In order to validate the procedure, we first expressed two previously characterized cyclic di-GMP binding proteins and their respective binding mutants, namely, the cellulose biosynthesis enhancer BcsE and its non-binding mutant BcsE R415D and the flagella motor "backstop-brake" protein YcgR and its non-binding mutant YcgR R118D in E. coli Top10. 4,15 Measurement of the cyclic di-GMP counts upon application of cell lysates expressing the cyclic di-GMP receptors and incubated with cyclic di-GMP showed higher counts derived from the membrane taken from the application spot than from the periphery. In contrast, cell lysates expressing the non-binding receptor variants and incubated with cyclic di-GMP had similar counts from the two membranes ( Figure 2A). Although independent experiments showed a similar trend, the absolute values and the ratio of counts from the application spot versus periphery membrane varied significantly.
Further optimization of the procedure will lead to more consistent outcome.
YciR, a GGDEF/EAL enzyme with opposing diguanylate cyclase and phosphodiesterase activities, has been initially characterized to downregulate expression of the agar-grown rdar (red, dry, and rough) biofilm morphotype in E. coli and Salmonella typhimurium. This mechanism is independent of its catalytic activities involving cyclic di-GMP sensing and protein-protein interactions. [16][17][18][19] Natural YciR protein variants encoded by semi-constitutive rdar biofilm expressing E. coli strains, though, failed to efficiently downregulate this morphotype, even when overexpressed. Sensing and binding of cyclic di-GMP might be a determinative factor for the different variant behavior. To assess whether two YciR protein variants differentially bind cyclic di-GMP, we choose to express YciR Fec101 , which has been previously shown to efficiently downregulate the rdar morphotype, and YciR TOB1 that is nearly invariant to modulation of rdar morphotype expression upon plasmid-based expression. 16  binding assay showed counts from the membrane periphery to be even higher than for the application spot suggesting that YciR TOB1 does not bind cyclic di-GMP. In contrast, assessment of binding of YciR Fec101 expressing cell extracts showed higher counts for the membrane derived from the application spot compared with the membrane from the periphery suggesting binding of cyclic di-GMP to YciR Fec101 .
We further assessed whether amino acid substitutions in the catalytic sites affect binding of cyclic di-GMP to YciR Fec101 . Thereby, we chose two protein variants with opposite consequences on rdar morphotype expression upon overexpression compared with the wild-type protein. In conclusion, a MALDI-TOF MS-based DRaCALA assay for cyclic di-nucleotide binding proteins was able to detect protein-ligand interactions. This MS-based assay has the potential to save experiment time, as production of radioactive cyclic di-GMP is not necessary. Future miniaturization of the experiment will save material.
The application of imaging MS will give a direct spatial assessment of ligand distribution and yield more reproducible results. Optimization of the experimental protocol and choice of a protein-binding matrix with optimal capillary forces for small compound diffusion in combination with an array-like experimental setup will enhance the detection process. Furthermore, the approach can be readily transferred to measure binding of other small ligands and molecules where methodological restrictions do not readily allow radioactive labeling. F I G U R E 3 Determination of the cyclic di-GMP binding capability of candidate receptor proteins by radioactive differential radial capillary action of ligand assay. 3,7 (A) Quantification of the fraction of 32 P-cyclic di-GMP bound to whole cell lysates of Escherichia coli Top10 containing the indicated plasmids. Mut1, YciR Fec101-D316A/ E317A/E440A cloned in pBAD30; Mut2, YciR Fec101-K581A cloned in pBAD30; pBAD30, vector control; pYciR Fec101, YciR Fec101 cloned in pBAD30; pYciR TOB1, YciR TOB1 cloned in pBAD30. (B, C) Ability of 250 μM cyclic di-GMP (cdiGMP), GTP, GMP, and cyclic GMP (cGMP) to interfere with 32 P-cyclic di-GMP binding in YciR Tob1 (B) and YciR Fec101 (C) containing lysates. No com, no competitor